AFFINITY MATURED CRIg VARIANTS

ABSTRACT

The present invention concerns affinity matured CRIg variants. In particular, the invention concerns CRIg variants having increased binding affinity to C3b and retaining selective binding to C3b over C3.

This application is a divisional application of U.S. patent applicationSer. No. 12/387,794, filed May 6, 2009, which application claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/189,653,filed Aug. 20, 2008 and U.S. Provisional Patent Application Ser. No.61/050,888, filed May 6, 2008, the disclosures of which are incorporatedhereby by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 24, 2015, isnamed GNE-0322 R1D1US_Sequence Listing.txt and is 45,972 bytes in size.

FIELD OF THE INVENTION

The present invention concerns affinity matured CRIg variants. Inparticular, the invention concerns CRIg variants having increasedbinding affinity to C3b and retaining selective binding to C3b over C3.

BACKGROUND OF THE INVENTION

The Complement System

The complement system is a complex enzyme cascade made up of a series ofserum glycoproteins that normally exist in inactive, pro-enzyme form.Three main pathways, the classical, alternative and mannose-bindinglectin pathway, can activate complement, which merge at the level of C3,where two similar C3 convertases cleave C3 into C3a and C3b.

Macrophages are specialist cells that have developed an innate capacityto recognize subtle differences in the structure of cell-surfaceexpressed identification tags, so called molecular patterns (Taylor, etal., Eur J Immunol 33, 2090-1097 (2003); Taylor, et al., Annu RevImmunol 23, 901-944 (2005)). While the direct recognition of thesesurface structures is a fundamental aspect of innate immunity,opsonization allows generic macrophage receptors to mediate engulfment,increasing the efficiency and diversifying recognition repertoire of thephagocyte (Stuart and Ezekowitz, Immunity 22, 539-550 (2005)). Theprocess of phagocytosis involves multiple ligand-receptor interactions,and it is now clear that various opsonins, including immunoglobulins,collectins, and complement components, guide the cellular activitiesrequired for pathogen internalization through interaction withmacrophage cell surface receptors (reviewed by Aderem and Underhill,Annu Rev Immunol 17, 593-623 (1999); Underhill and Ozinsky, Annu RevImmunol 20, 825-852 (2002)). While natural immunoglobulins encoded bygermline genes can recognize a wide variety of pathogens, the majorityof opsonizing IgG is generated through adaptive immunity, and thereforeefficient clearance through Fc receptors is not immediate (Carroll, NatImmunol 5, 981-986 (2004)). Complement, on the other hand, rapidlyrecognizes pathogen surface molecules and primes the particle for uptakeby complement receptors (Brown, Infect Agents Dis 1, 63-70 (1991)).

Complement consists of over 30 serum proteins that opsonize a widevariety of pathogens for recognition by complement receptors. Dependingon the initial trigger of the cascade, three pathways can bedistinguished (reviewed by (Walport, N Engl J Med 344, 1058-1066(2001)). All three share the common step of activating the centralcomponent C3, but they differ according to the nature of recognition andthe initial biochemical steps leading to C3 activation. The classicalpathway is activated by antibodies bound to the pathogen surface, whichin turn bind the C1q complement component, setting off a serine proteasecascade that ultimately cleaves C3 to its active form, C3b. The lectinpathway is activated after recognition of carbohydrate motifs by lectinproteins. To date, three members of this pathway have been identified:the mannose-binding lectins (MBL), the SIGN-R1 family of lectins and theficolins (Pyz et al., Ann Med 38, 242-251 (2006)) Both MBL and ficolinsare associated with serine proteases, which act like C1 in the classicalpathway, activating components C2 C4 leading to the central C3 step. Thealternative pathway contrasts with both the classical and lectinpathways in that it is activated due to direct reaction of the internalC3 ester with recognition motifs on the pathogen surface. Initial C3binding to an activating surface leads to rapid amplification of C3bdeposition through the action of the alternative pathway proteasesFactor B and Factor D. Importantly, C3b deposited by either theclassical or the lectin pathway also can lead to amplification of C3bdeposition through the actions of Factors B and D. In all three pathwaysof complement activation, the pivotal step in opsonization is conversionof the component C3 to C3b. Cleavage of C3 by enzymes of the complementcascades exposes the thioester to nucleophilic attack, allowing covalentattachment of C3b onto antigen surfaces via the thioester domain. Thisis the initial step in complement opsonization. Subsequent proteolysisof the bound C3b produces iC3b, C3c and C3dg, fragments that arerecognized by different receptors (Ross and Medof, Adv Immunol 37,217-267 (1985)). This cleavage abolishes the ability of C3b to furtheramplify C3b deposition and activate the late components of thecomplement cascade, including the membrane attack complex, capable ofdirect membrane damage. However, macrophage phagocytic receptorsrecognize C3b and its fragments preferentially; due to the versatilityof the ester-bond formation, C3-mediated opsonization is central topathogen recognition (Holers et al., Immunol Today 13, 231-236 (1992)),and receptors for the various C3 degradation products therefore play animportant role in the host immune response.

C3 itself is a complex and flexible protein consisting of 13 distinctdomains. The core of the molecule is made up of 8 so-calledmacroglobulin (MG) domains, which constitute the tightly packed α and βchains of C3. Inserted into this structure are CUB (C1r/C1s, Uegf andBone mophogenetic protein-1) and TED domains, the latter containing thethioester bond that allows covalent association of C3b with pathogensurfaces. The remaining domains contain C3a or act as linkers andspacers of the core domains. Comparison of C3b and C3c structures to C3demonstrate that the molecule undergoes major conformationalrearrangements with each proteolysis, which exposes not only the TED,but additional new surfaces of the molecule that can interact withcellular receptors (Janssen and Gros, Mol Immunol 44, 3-10 (2007)).

Complement C3 Receptors on Phagocytic Cells

There are three known gene superfamilies of complement receptors: Theshort consensus repeat (SCR) modules that code for CR1 and CR2, thebeta-2 integrin family members CR3 and CR4, and the immunoglobulinIg-superfamily member CRIg.

CR1 is a 180-210 kDa glycoprotein consisting of 30 Short ConsensusRepeats (SCRs) and plays a major role in immune complex clearance. SCRsare modular structures of about 60 amino acids, each with two pairs ofdisulfide bonds providing structural rigidity. High affinity binding toboth C3b and C4b occurs through two distinct sites, each composed of 3SCRs)reviewed by (Krych-Goldberg and Atkinson, Immunol Rev 180, 112-122(2001)). The structure of the C3b binding site, contained within SCR15-17 of CR1 (site 2), has been determined by MRI (Smith et al., Cell108, 769-780 (2002)), revealing that the three modules are in anextended head-to-tail arrangement with flexibility at the 16-17junction. Structure-guided mutagenesis identified a positively chargedsurface region on module 15 that is critical for C4b binding. Thispatch, together with basic side chains of module 16 exposed on the sameface of CR1, is required for C3b binding. The main function of CR1,first described as an immune adherence receptor (Rothman et al., JImmunol 115, 1312-1315 (1975)), is to capture ICs on erythrocytes fortransport and clearance by the liver (Taylor et al., Clin ImmunolImmunopathol 82, 49-59 (1997)). There is a role in phagocytosis for CR1on neutrophils, but not in tissue macrophages (Sengelov et al., JImmunol 153, 804-810 (1994)). In addition to its role in clearance ofimmune complexes, CR1 is a potent inhibitor of both classical andalternative pathway activation through its interaction with therespective convertases (Krych-Goldberg and Atkinson, 2001, supra;Krych-Goldberg et al., J Biol Chem 274, 31160-31168 (1999)). In themouse, CR1 and CR2 are two products of the same gene formed byalternative splicing and are primarily associated with B-lymphocytes andfollicular dendritic cells and function mainly in regulating B-cellresponses (Molina et al., 1996). The mouse functional equivalent of CR1,Crry, inactivates the classical and alternative pathway enzymes and actsas an intrinsic regulator of complement activation rather than as aphagocytic receptor (Molina et al., Proc Natl Acad Sci USA 93, 3357-3361(1992)).

CR2 (CD21) binds iC3b and C3dg and is the principal complement receptorthat enhances B cell immunity (Carroll, Nat Immunol 5, 981-986 (2004);Weis et al., Proc Natl Acad Sci USA 81, 881-885 (1984)). Uptake ofC3d-coated antigen by cognate B cells results in an enhanced signal viathe B cell antigen receptor. Thus, coengagement of the CD21-CD19-CD81coreceptor with B cell antigen receptor lowers the threshold of B cellactivation and provides an important survival signal (Matsumoto et al.,J Exp Med 173, 55-64 (1991)). The CR2 binding site on iC3b has beenmapped partly on the interface between the TED and the MG1 domains(Clemenza and Isenman, J Immunol 165, 3839-3848 (2000)).

CR3 and CR4 are transmembrane heterodimers composed of an alpha subunit(CD11b or α_(M) and CD11c or α_(X), respectively) and a common betachain (CD18 or β₂), and are involved in adhesion to extracellular matrixand to other cells as well as in recognition of iC3b. They belong to theintegrin family and perform functions not only in phagocytosis, but alsoin leukocyte trafficking and migration, synapse formation andcostimulation (reviewed by (Ross, Adv Immunol 37, 217-267 (2000)).Integrin adhesiveness is regulated through a process called inside-outsignaling, transforming the integrins from a low- to a high-affinitybinding state (Liddington and Ginsberg, J Cell Biol 158, 833-839(2002)). In addition, ligand binding transduces signals from theextracellular domain to the cytoplasm. The binding sites of iC3b havebeen mapped to several domains on the alpha chain of CR3 and CR4(Diamond et al., J Cell Biol 120, 1031-1043 (1993); Li and Zhang, J BiolChem 278, 34395-34402 (2003); Xiong and Zhang, J Biol Chem 278,34395-34402 (2001)). The multiple ligands for CR3: iC3b, beta-glucan andICAM-1, seem to bind to partially overlapping sites contained within theI domain of CD11b (Balsam et al., 1998; Diamond et al., 1990; Zhang andPlow, 1996). Its specific recognition of the proteolytically inactivatedform of C3b, iC3b, is predicted based on structural studies that locatethe CR3 binding sites to residues that become exposed upon unfolding ofthe CUB domain in C3b (Nishida et al., Proc Natl Acad Sci USA 103,19737-19742 (2006)), which occurs upon α chain cleavage by thecomplement regulatory protease, Factor I.

CRIg is a macrophage associated receptor with homology to A33 antigenand JAM1 that is required for the clearance of pathogens from the bloodstream. A human CRIg protein was first cloned from a human fetal cDNAlibrary using degenerate primers recognizing conserved Ig domains ofhuman JAM1. Sequencing of several clones revealed an open reading frameof 400 amino acids. Blast searches confirmed similarity to Z39Ig, a type1 transmembrane protein (Langnaese et al., Biochim Biophys Acta 1492(2000) 522-525). The extracellular region of this molecule was found toconsist of two Ig-like domains, comprising an N-terminal V-set domainand a C-terminal C2-set domain. The novel human protein was originallydesignated as a “single transmembrane Ig superfamily member macrophageassociated” (huSTIgMA). (huSTIgMA). Subsequently, using 3′ and 5′primers, a splice variant of huSTIgMA was cloned, which lacks themembrane proximal IgC domain and is 50 amino acids shorter. Accordingly,the shorter splice variant of this human protein was designatedhuSTIgMAshort. The amino acid sequence of huSTIgMA (referred to asPRO362) and the encoding polynucleotide sequence are disclosed in U.S.Pat. No. 6,410,708, issued Jun. 25, 2002. In addition, both huSTIgMA andhuSTIgMAshort, along with the murine STIgMA (muSTIgMA) protein andnucleic acid sequences, are disclosed in PCT Publication WO 2004031105,published Apr. 15, 2004.

The crystal structure of CRIg and a C3b:CRIg complex is disclosed inU.S. Application Publication No. 2008/0045697, published Feb. 21, 2008.

The Kupffer cells (KCs), residing within the lumen of the liversinusoids, form the largest population of macrophages in the body.Although KCs have markers in common with other tissue residentmacrophages, they perform specialized functions geared towards efficientclearance of gut-derived bacteria, microbial debris, bacterialendotoxins, immune complexes and dead cells present in portal vein blooddraining from the microvascular system of the digestive tract (Bilzer etal., Liver Int 26, 1175-1186 (2006)). Efficient binding of pathogens tothe KC surface is a crucial step in the first-line immune defenseagainst pathogens (Benacerraf et al., J Exp Med 110, 27-48 (1959)). Acentral role for KCs in the rapid clearance of pathogens from thecirculation is illustrated by the significantly increased mortality inmice depleted of KCs (Hirakata et al., Infect Immun 59, 289-294 (1991)).The identification of CRIg further stresses the critical role ofcomplement and KCs in the first line immune defense against circulatingpathogens.

The only complement C3 receptors identified on mouse KCs are CRIg andCR3 (Helmy et al., Cell 124, 915-927 (2006)), while human KCs showadditional expression of CR1 and CR4 (Hinglais et al., 1989). Both CRIgand CR3 on KCs contribute to binding to iC3b opsonized particles invitro (Helmy et al., Lab Invest 61, 509-514 (2006)). In vivo, a role ofKC-expressed CR3 in the binding to iC3b-coated pathogens is less clear.CR3 has been proposed to contribute to clearance of pathogens indirectlyvia recruitment of neutrophils and interaction with neutrophil-expressedICAM1 (Conlan and North, Exp Med 179, 259-268 (1994); Ebe et al., PatholInt 49, 519-532 (1999); Gregory et al., J Immunol 157, 2514-2520 (1996);Gregory and Wing, J Leukoc Biol 72, 239-248 (2002); Rogers and Unanue,Infect Immun 61, 5090-5096 (1993)). In contrast, CRIg performs a directrole by capturing pathogens that transit through the liver sinusoidallumen (Helmy et al., 2006, supra). A difference in the biology of CRIgvs CR3 is in part reflected by difference in binding characteristics ofthese two receptors. CRIg expressed on KCs constitutively binds tomonomeric C3 fragments whereas CR3 only binds to iC3b-opsonizedparticles (Helmy et al., 2006, supra). The capacity of CRIg toefficiently capture monomeric C3b and iC3b as well as C3b/iC3b-coatedparticles reflects the increased avidity created by a multivalentinteraction between CRIg molecules concentrated at the tip of membraneextensions of macrophages (Helmy et al., 2006, supra) and multimers ofC3b and iC3b present on the pathogen surface. While CR3 only bindsiC3b-coated particles, CRIg additionally bind to C3b, the first C3cleavage product formed on serum-opsonized pathogens (Croize et al.,Infect Immun 61, 5134-5139 (1993)). Since a large number of C3bmolecules bound to the pathogen surface are protected from cleavage byfactor H and I (Gordon et al., J Infect Dis 157, 697-704 (1988)),recognition of C3b ligands by CRIg ensures rapid binding and clearance.Thus, while both CRIg and CR3 are expressed on KCs, they show differentligand specificity, distinct binding properties and distinct kinetics ofpathogen clearance.

Examples of pathogens that exploit cell surface receptors for cellularentry are viruses like human immunodeficiency virus (HIV), andintracellular bacteria like Mycobacterium tuberculosum, Mycobacteriumleprae, Yersinia pseudotuberculosis, Salmonella typhimurium and ListeriaMonocytogenes and parasites like the prostigmatoid Leishmania major(Cossart and Sansonetti, Science 304:242-248 (2004); Galan, Cell103:363-366 (2000); Hornef et al., Nat. Immunol. 3:1033-1040 (2002);Stoiber et al., Mol. Immunol. 42:153-160 (2005)).

As discussed above, CRIg is a recently discovered complement C3 receptorexpressed on a subpopulation of tissue resident macrophages. Next tofunctioning as a complement receptor for C3 proteins, the extracellularIgV domain of CRIg selectively inhibits the alternative pathway ofcomplement by binding to C3b and inhibiting proteolytic activation of C3and C5. However, CRIg binding affinity for the convertase subunit C3b islow (IC50>1 μM) requiring a relatively high concentration of protein toreach near complete complement inhibition. Accordingly, there is a needfor CRIg polypeptides with improved therapeutic efficacy. The presentinvention provides such polypeptides.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the construction ofa CRIg variant with enhanced binding affinity. A CRIg-ECD protein withcombined amino acid substitutions Q64R and M86Y showed a 30 foldincreased binding affinity and a 7 fold improved complement inhibitoryactivity over the wildtype CRIg variant. In addition, treatment with theaffinity-improved CRIg fusion protein in a mouse model of arthritisresulted in a significant reduction in clinical scores compared totreatment with a wild-type CRIg protein

Accordingly, the present invention concerns CRIg variants.

In one aspect, the invention concerns a CRIg variant comprising an aminoacid substitution in a region selected from the group consisting ofE8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111 of the amino acidsequence of SEQ ID NO: 2.

In one embodiment, the variant selectively binds to C3b over C3, or afragment thereof.

In another embodiment, the variant has increased binding affinity to C3bover native sequence human CRIg of SEQ ID NO: 2, where the bindingaffinity may, for example, be increased by at lest 2 fold, or by atleast 3 fold, or by at least 4 fold, or by at least 5 fold, or by atleast 6 fold, or by at least 7 fold, or by at least 9 fold, or by atleast 10 fold, or by at least 15 fold, or by at least 20 fold, or by atleast 30 fold, or by at least 40 fold, or by at least 50 fold, or by atleast 70 fold, or by at least 80 fold, or by at least 90 fold, or by atleast 100 fold.

In yet another embodiment, the variant is a more potent inhibitor of thealternative complement pathway than native sequence human CRIg of SEQ IDNO: 2.

In a further embodiment, the variant comprises an amino acidsubstitution at one or more amino acid positions selected from the groupconsisting of positions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105, and110 in the amino acid sequence of SEQ ID NO: 2.

In a still further embodiment, the variant comprises an amino acidsubstitution at one or more of amino acid positions 60, 64, 86, 99, 105and 110 in the amino acid sequence of SEQ ID NO: 2.

In an additional embodiment, the variant comprises one or moresubstitutions selected from the group consisting of E8W, W14F,E84Y/W14F; P45F; G42D/D44H/P45F; Q60I; Q64R; Q60I/Q64R; M86Y; M86W,M86F, M86W/Q9R; M86F/Q99R; K110D, K11N; Q105R/K110N; Q105R/K110Q; andQ105K/K110D.

In another embodiment, the variant comprises one or more substitutionsselected from the group consisting of Q64R/M86Y; Q60I/Q64R/E8Y;Q60I/Q64R/G42D; Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F;Q60I/Q64R/M86Y; Q60I/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N;Q60I/Q105R/K110N; M86Y/E8Y; M86Y/G42D/D44H/P45F; M86Y/P45F;M86Y/G42D/D44H/P45F; andM86Y/Q99K/M86Y/Q99R/M86Y/Q105R/M86Y/Q105K/M86Y/Q105R/K110N.

In yet another embodiment, the variant comprises one or moresubstitutions selected from the group consisting of Q60I; Q64R;Q60I/Q64R; M86Y; Q99L; Q105K/K110D; E8W/Q105R/K110N; Q64R/M86Y;Q60I/Q64R/E8Y; Q60I/Q64R/G42D; Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F;Q60I/Q64R/M86Y; Q601/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N;M86Y/P45F; and M86Y/Q105K.

In a more specific embodiment, the variant comprises a Q60I/Q64R/M86Y orQ60I/Q64R/G42D/D44H/P45F substitution.

In another aspect, the invention concerns a chimeric comprising a CRIgvariant as defined herein.

In one embodiment, the chimeric molecule is an immunoadhesin.

In another embodiment, the immunoadhesin comprises a CRIg variant thatis shorter than the full-length CRIg of SEQ ID NO: 2.

In yet another embodiment, the chimeric molecule comprises a CRIgextracellular domain.

In a further aspect, the invention concerns a pharmaceutical compositioncomprising a CRIg variant or a chimeric molecule, e.g. an immunoadhesinof the present invention, in admixture with a pharmaceuticallyacceptable excipient.

In a still further aspect, the invention concerns a method for theprevention or treatment of a complement-associated disease or condition,comprising administering to a subject in need of such treatment aprophylactically or therapeutically effective amount of a CRIg variantor a chimeric molecule, such as an immunoadhesin, comprising suchvariant.

In one embodiment, the complement-associated disease is an inflammatorydisease or an autoimmune disease.

In another embodiment, the complement-associated disease is selectedfrom the group consisting of rheumatoid arthritis (RA), adultrespiratory distress syndrome (ARDS), remote tissue injury afterischemia and reperfusion, complement activation during cardiopulmonarybypass surgery, dermatomyositis, pemphigus, lupus nephritis andresultant glomerulonephritis and vasculitis, cardiopulmonary bypass,cardioplegia-induced coronary endothelial dysfunction, type IImembranoproliferative glomerulonephritis, IgA nephropathy, acute renalfailure, cryoglobulemia, antiphospholipid syndrome, age-related maculardegeneration, uveitis, diabetic retinopathy, allo-transplantation,hyperacute rejection, hemodialysis, chronic occlusive pulmonary distresssyndrome (COPD), asthma, aspiration pneumonia, utricaria, chronicidiopathic utricaria, hemolytic uremic syndrome, endometriosis,cardiogenic shock, ischemia reperfusion injury, and multiple schlerosis(MS).

In yet another embodiment, the complement-associated disease is selectedfrom the group consisting of inflammatory bowel disease (IBD), systemiclupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis,spondyloarthropathies, systemic sclerosis (scleroderma), idiopathicinflammatory myopathies (dermatomyositis, polymyositis), Sjogren'ssyndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic anemia(immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other nonhepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory and fibrotic lungdiseases (e.g., cystic fibrosis), gluten-sensitive enteropathy,Whipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases of the lung such as eosinophilic pneumonia,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis,transplantation associated diseases including graft rejection,graft-versus host disease, Alzheimer's disease, paroxysmal nocturnalhemoglobinurea, hereditary angioedema, atherosclerosis and type IImembranoproliferative glomerulonephritis.

In a preferred embodiment, the complement-associated disease isrheumatoid arthritis (RA).

In another preferred embodiment, the complement-associated disease is acomplement-associated eye condition.

In a further embodiment, the complement-associated eye condition isselected from the group consisting of all stages of age-related maculardegeneration (AMD), uveitis, diabetic and other ischemia-relatedretinopathies, endophthalmitis, and other intraocular neovasculardiseases.

In a still further embodiment, the intraocular neovascular disease isselected from the group consisting of diabetic macular edema,pathological myopia, von Hippel-Lindau disease, histoplasmosis of theeye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization,and retinal neovascularization.

In yet another embodiment, the complement-associated eye condition isselected from the group consisting of age-related macular degeneration(AMD), choroidal neovascularization (CNV), diabetic retinopathy (DR),and endophthalmitis, where AMD includes both wet and dry or atrophicAMD.

In one embodiment, the patient is a mammal, preferable a human.

In another aspect, the invention concerns a method for inhibition of theproduction of C3b complement fragment in a mammal comprisingadministering to said mammal an effective amount of a CRIg variant ofthe present invention, or an immunoadhesin comprising such variant.

In yet another aspect, the invention concerns a method for selectiveinhibition of the alternative complement pathway in a mammal, comprisingadministering to said mammal an effective amount of a CRIg variant ofthe present invention, or an immunoadhesin comprising such variant.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1B show the nucleotide and amino acid sequences of the399-amino acid full-length long form of native human CRIg (huCRIg, SEQID NOS: 1 and 2, respectively).

FIGS. 2A-2B show the nucleotide and amino acid sequences of the305-amino acid short form of native human CRIg (huCRIg-short, SEQ IDNOS: 3 and 4, respectively).

FIGS. 3A-3C show the nucleotide and amino acid sequences of the280-amino acid native murine CRIg (muCRIg, SEQ ID NOS: 5 and 6,respectively).

FIG. 4: Activity of CRIg mutants in binding assay and inhibition assay.Binding affinity for CRIg was measured as competitive displacement ofC3b (FIG. 4A), and the biological activity was measured by a hemolysisinhibition assay. PUR10680 was wild-type control (red), RIL 41 (blue)and RL41 (green) were two mutants (FIG. 4B). (FIG. 4C) Stepwiseoptimization of the CRIg binding interface.

FIG. 5: Correlation between competitive ELISA and hemolytic assay.

FIGS. 6A-B: CRIg mutant Q64R/M86Y shows improved binding affinity byBiacore analysis. (FIG. 6A) SPR sensograms generated by injection ofincreasing concentrations of C3b over coated CRIg wt and CRIg Q64R M86Yproteins. FIG. 6B. Steady state analysis of the binding data indicates aKd of 0.2 micromolar for the Q64R/M86Y mutant and 1.1 micromolar forwild-type CRIg.

FIG. 7: Affinity-improved CRIg remains selective for C3b. Alpha Screencompetitive assay was utilized on purified C3 and C3b.

FIGS. 8A-B: Improved complement inhibitory potency of CRIg Q64R M86Ycompared to wildtype CRIg. (FIG. 8A) Complement inhibition by wild-typeCRIg and CRIg Q46R M86Y were compared using an alternativepathway-selective hemolytic assay using rabbit red blood cells and C 1q-depleted human serum. (FIG. 8B) Complement inhibition by wild-typeCRIg and CRIg Q46R M86Y were compared using an ELISA-based alternativepathway assay with microwell plate-coated LPS and C1q-depleted humanserum.

FIGS. 9A-D: CRIg Q64R M86Y shows improved efficacy in vivo over CRIg WT.

(FIG. 9A) Clinical scores of mice injected with KRN serum and treatedwith various concentrations and versions of wild-type andaffinity-matured recombinant human and mouse CRIg proteins. Datarepresent mean of 4-7 mice per group. (FIG. 9B) Scatter plots ofclinical scores from individual mice at day 6 following serum transfer.(FIG. 9C) Hematoxylin and eosin-stained sections of mice treated withCRIg wt of CRIg Q64R M86Y 6 days following serum transfer. (FIG. 9D)Scatter plots of histological scores from mice treated with CRIg wt orCRIg Q64R M86Y 6 days following serum transfer.

FIG. 10: Phage libraries. Five soft-randomized libraries were designedto cover the contact area between CRIg and C3b.

FIG. 11: Step-wise generation of higher affinity CRIg my phage display.Selected mutants of CRIg anti-C3b from the five soft-randomizedlibraries. Each panel shows clones that were selected from each librarybased on binding affinity to C3b. The sequence is denoted by thesingle-letter amino acid code. Each panel compares the individualmutants with the consensus and parent wild-type (WT) sequences. FIG. 11discloses SEQ ID NOS 21-63 and 63-67, respectively, in order ofappearance.

FIG. 12: Comparison of binding affinities, determined by competitiveELISA, and in vivo hemolysis inhibition for selected mutants. Mutantswith a greater than 5 fold increased in binding affinity or in vivopotency are shaded yellow.

FIG. 13: Comparison of binding affinity and in vivo hemolysis inhibitionfor second generation mutants (parent sequences shown in gray). Mutantswith a greater than 5 fold increase over the parent mutant in bindingaffinity are highlighted in blue, mutants with a greater than 90 foldincrease in binding affinity are highlighted in yellow. Similarly,mutants with greater in vivo potency than parent sequences arehighlighted in orange.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms “CRIg,” “PRO362,” “JAM4,” and “STIgMA” are usedinterchangeably, and refer to native sequence and variant CRIgpolypeptides.

A “native sequence” CRIg, is a polypeptide having the same amino acidsequence as a CRIg polypeptide derived from nature, regardless of itsmode of preparation. Thus, native sequence CRIg can be isolated fromnature or can be produced by recombinant and/or synthetic means. Theterm “native sequence CRIg”, specifically encompassesnaturally-occurring truncated or secreted forms of CRIg (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofCRIg. Native sequence CRIg polypeptides specifically include thefull-length 399 amino acids long human CRIg polypeptide of SEQ ID NO: 2(huCRIg, shown in FIGS. 1A and 1B), with or without an N-terminal signalsequence, with or without the initiating methionine at position 1, andwith or without any or all of the transmembrane domain at about aminoacid positions 277 to 307 of SEQ ID NO: 2. In a further embodiment, thenative sequence CRIg polypeptide is the 305-amino acid, short form ofhuman CRIg (huCRIg-short, SEQ ID NO: 4, shown in FIGS. 2A and 2B), withor without an N-terminal signal sequence, with or without the initiatingmethionine at position 1, and with or without any or all of thetransmembrane domain at about positions 183 to 213 of SEQ ID NO: 4. In adifferent embodiment, the native sequence CRIg polypeptide is a 280amino acids long, full-length murine CRIg polypeptide of SEQ ID NO: 6(muCRIg, shown in FIGS. 3A-3C), with or without an N-terminal signalsequence, with or without the initiating methionine at position 1, andwith or without any or all of the transmembrane domain at about aminoacid positions 181 to 211 of SEQ ID NO: 6. CRIg polypeptides of othernon-human animals, including higher primates and mammals, arespecifically included within this definition.

The CRIg “extracellular domain” or “ECD” refers to a form of the CRIgpolypeptide, which is essentially free of the transmembrane andcytoplasmic domains of the respective full length molecules. Ordinarily,the CRIg ECD will have less than 1% of such transmembrane and/orcytoplasmic domains and preferably, will have less than 0.5% of suchdomains. CRIg ECD may comprise amino acid residues 1 or about 21 to X ofSEQ ID NO: 2, 4, or 6, where X is any amino acid from about 271 to 281in SEQ ID NO: 2, any amino acid from about 178 to 186 in SEQ ID NO: 4,and any amino acid from about 176 to 184 in SEQ ID NO: 6.

The term “CRIg variant,” as used herein, means an active CRIgpolypeptide as defined below having at least about 80% amino acidsequence identity to a native sequence CRIg polypeptide, including,without limitation, the full-length huCRIg (SEQ ID NO: 2), huCRIg-short(SEQ ID NO: 4), and muCRIg (SEQ ID NO: 6), each with or without theN-terminal initiating methionine, with or without the N-terminal signalsequence, with or without all or part of the transmembrane domain andwith or without the intracellular domain. In a particular embodiment,the CRIg variant has at least about 80% amino acid sequence homologywith the mature, full-length polypeptide from within the sequence of thesequence of SEQ ID NO: 2. In another embodiment, the CRIg variant has atleast about 80% amino acid sequence homology with the mature,full-length polypeptide from within the sequence of SEQ ID NO: 4. In yetanother embodiment, the CRIg variant has at least about 80% amino acidsequence homology with the mature, full-length polypeptide from withinthe sequence of SEQ ID NO: 6. Ordinarily, a CRIg variant will have atleast about 80% amino acid sequence identity, or at least about 85%amino acid sequence identity, or at least about 90% amino acid sequenceidentity, or at least about 95% amino acid sequence identity, or atleast about 98% amino acid sequence identity, or at least about 99%amino acid sequence identity with the mature amino acid sequence fromwithin SEQ ID NO: 2, 4, or 6. Throughout the description, including theexamples, the term “wild-type” or “WT” refers to the mature full-lengthshort form of human CRIg (CRIg(S)) (SEQ ID NO: 4), and the numbering ofamino acid residues in the CRIg variants refers to the sequence of SEQID NO: 4

The CRIg variants of the present invention are CRIg agonists, ashereinafter defined. In particular, the CRIg variants herein maintainselective binding to C3b over C3, where “selective binding” is used torefer to binding to C3b and a lack of binding to C3. In addition, in apreferred embodiment, the CRIg variants of the present invention haveincreased binding affinity to C3b relative to a native sequence CRIgpolypeptide, such as the human long form of CRIg (SEQ ID NO: 2). Invarious embodiments, the increase in binding affinity is at least about2 fold, or at least about 3 fold, or at least about 4 fold, or at leastabout 5 fold, or at least about 6 fold, or at least about 7 fold, or atleast about 8 fold, or at least about 9 fold, or at least about 10 fold,or at least about 15 fold, or at least about 20 fold, or at least about25 fold, or at least about 30 fold, or at least about 35 fold, or atleast about 40 fold, or at least about 45 fold, or at least about 50fold, or at least about 55 fold, or at least about 60 fold, or at leastabout 65 fold, or at least about 70 fold, or at least about 75 fold, orat least about 80 fold, or at least about 85 fold, or at least about 90fold, or at least about 95 fold, or at least about 100 fold, relative tothe native sequence human CRIg polypeptide of SEQ ID NO: 2. In otherembodiments, the increase in binding affinity to C3b relative to thenative sequence human CRIg polypeptide of SEQ ID NO: 2 is about 5-10fold, or about 5-15 fold, or about 5-20 fold, or about 5-25 fold, orabout 5-25 fold, or about 5-30 fold, or about 5-35 fold, or about 5-40fold, or about 5-45 fold, or about 5-50 fold, or about 5-55 fold, orabout 5-60 fold, or about 5-65 fold, or about 5-70 fold, or about 5-75fold, or about 5-80 fold, or about 5-85 fold, or about 5-90 fold, orabout 5-95 fold, or about 5-100 fold.

“Percent (%) amino acid sequence identity” with respect to the CRIgvariants herein is defined as the percentage of amino acid residues in aCRIg variant sequence that are identical with the amino acid residues inthe native CRIg sequence to which they are compared, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. For sequences thatdiffer in length, percent sequence identity is determined relative tothe longer sequence, along the full length of the longer sequences.Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. Sequence identity is thencalculated relative to the longer sequence, i.e. even if a shortersequence shows 100% sequence identity with a portion of a longersequence, the overall sequence identity will be less than 100%.

“Percent (%) nucleic acid sequence identity” with respect to the CRIgvariant encoding sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the CRIg variant encoding sequence,respectively, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. Sequence identity is then calculatedrelative to the longer sequence, i.e. even if a shorter sequence shows100% sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

Included in the definition of a CRIg variant are all amino acid sequencevariants, as hereinabove defined, regardless of their mode ofidentification or preparation. Specifically included herein are variantsthat have been modified by substitution, chemically, enzymatically, orby other appropriate means with a moiety other than a naturallyoccurring amino acid, as long as they retain a qualitative biologicalproperty of a native sequence CRIg. Exemplary non-naturally occurringamino acid substitution include those described herein below.

Amino acid residues are classified into four major groups:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous solution.

Basic: The residue has a positive charge due to association with H ionat physiological pH and the residue is attracted by aqueous solution soas to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium atphysiological pH.

Neutral/non-polar: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. These residues are also designated“hydrophobic residues.”

Neutral/polar: The residues are not charged at physiological pH, but theresidue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

Amino acid residues can be further classified as cyclic or non-cyclic,aromatic or non aromatic with respect to their side chain groups thesedesignations being commonplace to the skilled artisan.

Commonly encountered amino acids which are not encoded by the geneticcode, include 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelicacid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, andother aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leuand other aliphatic amino acids; 2-aminoisobutyric acid (Aib) for Gly;cyclohexylalanine (Cha) for Val, and Leu and Ile; homoarginine (Har) forArg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His;N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylglycine (EtGly) forGly, Pro, and Ala; N-ethylasparigine (EtAsn) for Asn, and Gln;Hydroxyllysine (Hyl) for Lys; allohydroxyllysine (AHyl) for Lys; 3-(and4)hydoxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr; allo-isoleucine(AIle) for Ile, Leu, and Val; .rho.-amidinophenylalanine for Ala;N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala;N-methylisoleucine (MeIle) for Ile; Norvaline (Nva) for Met and otheraliphatic amino acids; Norleucine (Nle) for Met and other aliphaticamino acids; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) andmethionine sulfoxide (MSO) for Thr, Asn and Gln; N-methylphenylalanine(MePhe), trimethylphenylalanine, halo (F, Cl, Br, and I)phenylalanine,triflourylphenylalanine, for Phe.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented.

“Ameliorate” as used herein, is defined herein as to make better orimprove.

The term “mammal” as used herein refers to any animal classified as amammal, including, without limitation, humans, non-human primates,domestic and farm animals, and zoo, sports or pet animals such horses,pigs, cattle, dogs, cats and ferrets, etc. In a preferred embodiment ofthe invention, the mammal is a higher primate, most preferably human.

The term “complement-associated disease” is used herein in the broadestsense and includes all diseases and pathological conditions thepathogenesis of which involves abnormalities of the activation of thecomplement system, such as, for example, complement deficiencies. Theterm specifically include diseases and pathological conditions thatbenefit from the inhibition of C3 convertase. The term additionallyincludes diseases and pathological conditions that benefit frominhibition, including selective inhibition, of the alternativecomplement pathway. Complement-associated diseases include, withoutlimitation, inflammatory diseases and autoimmune diseases, such as, forexample, rheumatoid arthritis (RA), acute respiratory distress syndrome(ARDS), remote tissue injury after ischemia and reperfusion, complementactivation during cardiopulmonary bypass surgery, dermatomyositis,pemphigus, lupus nephritis and resultant glomerulonephritis andvasculitis, cardiopulmonary bypass, cardioplegia-induced coronaryendothelial dysfunction, type II membranoproliferativeglomerulonephritis, IgA nephropathy, acute renal failure,cryoglobulemia, antiphospholipid syndrome, age-related maculardegeneration, uveitis, diabetic retinopathy, allo-transplantation,hyperacute rejection, hemodialysis, chronic occlusive pulmonary distresssyndrome (COPD), asthma, and aspiration pneumonia. In a preferredembodiment, the “complement-associated disease” is a disease in whichthe alternative pathway of complement plays a prominent role, includingrheumatoid arthritis (RA), complement-associated eye conditions, such asage-related macular degeneration, anti-phospholipid syndrome, intestinaland renal ischemia-reperfusion injury, and type II membranoproliferativeglomerulonephritis.

The term “complement-associated eye condition” is used herein in thebroadest sense and includes all eye conditions and diseases thepathology of which involves complement, including the classical and thealternative pathways, and in particular the alternative pathway ofcomplement. Specifically included within this group are all eyeconditions and diseases the associated with the alternative pathway, theoccurrence, development, or progression of which can be controlled bythe inhibition of the alternative pathway. Complement-associated eyeconditions include, without limitation, macular degenerative diseases,such as all stages of age-related macular degeneration (AMD), includingdry and wet (non-exudative and exudative) forms, choroidalneovascularization (CNV), uveitis, diabetic and other ischemia-relatedretinopathies, endophthalmitis, and other intraocular neovasculardiseases, such as diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, Central Retinal VeinOcclusion (CRVO), corneal neovascularization, and retinalneovascularization. A preferred group of complement-associated eyeconditions includes age-related macular degeneration (AMD), includingnon-exudative (wet) and exudative (dry or atrophic) AMD, choroidalneovascularization (CNV), diabetic retinopathy (DR), andendophthalmitis.

The term “inflammatory disease” and “inflammatory disorder” are usedinterchangeably and mean a disease or disorder in which a component ofthe immune system of a mammal causes, mediates or otherwise contributesto an inflammatory response contributing to morbidity in the mammal.Also included are diseases in which reduction of the inflammatoryresponse has an ameliorative effect on progression of the disease.Included within this term are immune-mediated inflammatory diseases,including autoimmune diseases.

The term “T-cell mediated” disease means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to morbidity in amammal. The T cell mediated disease may be associated with cell mediatedeffects, lymphokine mediated effects, etc. and even effects associatedwith B cells if the B cells are stimulated, for example, by thelymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areT cell mediated, include, without limitation, inflammatory bowel disease(IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenilechronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic polyneuropathy, hepatobiliarydiseases such as infectious hepatitis (hepatitis A, B, C, D, E and othernonhepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases (e.g., cystic fibrosis),gluten-sensitive enteropathy, Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases of thelung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection, graft-versus host disease, Alzheimer'sdisease, and atherosclerosis.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Active” or “activity” in the context of variants of the CRIgpolypeptides of the invention refers to form(s) of such polypeptideswhich retain the biological and/or immunological activities of a nativeor naturally-occurring polypeptide of the invention. A preferredbiological activity is the ability to bind C3b, and/or to affectcomplement or complement activation, in particular to inhibit thealternative complement pathway and/or C3 convertase Inhibition of C3convertase can, for example, be measured by measuring the inhibition ofC3 turnover in normal serum during collagen- or antibody-inducedarthritis, or inhibition of C3 deposition is arthritic joints.

“Biological activity” in the context of a polypeptide that mimics CRIgbiological activity refers, in part, to the ability of such molecules tobind C3b and/or to affect complement or complement activation, inparticular, to inhibit the alternative complement pathway and/or C3convertase.

The term CRIg “agonist” is used in the broadest sense, and includes anymolecule that mimics a qualitative biological activity (as hereinabovedefined) of a native sequence CRIg polypeptide.

“Operably linked” refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence “operablylinked” to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequencesand wherein the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, then synthetic oligonucleotide adaptors orlinkers are used in accord with conventional practice.

“Control sequences” refer to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

“Expression system” refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, “cell,” “cell line,” and “cell culture” are usedinterchangeably and all such designations include progeny. Thus,“transformants” or “transformed cells” includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content because deliberate or inadvertent mutations mayoccur. Mutant progeny that have the same functionality as screened forin the originally transformed cell are included. Where distinctdesignations are intended, it will be clear from the context.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

A “phage display library” is a protein expression library that expressesa collection of cloned protein sequences as fusions with a phage coatprotein. Thus, the phrase “phage display library” refers herein to acollection of phage (e.g., filamentous phage) wherein the phage expressan external (typically heterologous) protein. The external protein isfree to. interact with (bind to) other moieties with which the phage arecontacted. Each phage displaying an external protein is a “member” ofthe phage display library.

The term “filamentous phage” refers to a viral particle capable ofdisplaying a heterogenous polypeptide on its surface, and includes,without limitation, fl, fd, Pfl, and M13. The filamentous phage maycontain a selectable marker such as tetracycline (e.g., “fd-tet”).Various filamentous phage display systems are well known to those ofskill in the art (see, e.g., Zacher et al., Gene, 9:127-140 (1980),Smith et al., Science, 228:1315-1317 (1985); and Parmley and Smith,Gene, 73:305-318 (1988)).

The term “panning” is used to refer to the multiple rounds of screeningprocess in identification and isolation of phages carrying compounds,such as antibodies, with high affinity and specificity to a target.

The phrase “conserved amino acid residues” is used to refer to aminoacid residues that are identical between two or more amino acidsequences aligned with each other.

II Detailed Description

Complement is an important component of the innate and adaptive immuneresponse, yet complement split products generated through activation ofeach of the three complement pathways (classical, alternative, andlectin) can cause inflammation and tissue destruction. Thus,uncontrolled complement activation due to the lack of appropriatecomplement regulation has been associated with various chronicinflammatory diseases. Dominant in this inflammatory cascade are thecomplement split products C3a and C5a that function as chemoattractantand activators of neutrophils and inflammatory macrophages via the C3aand C5a; receptors (Mollnes, T. E., W. C. Song, and J. D. Lambris. 2002.Complement in inflammatory tissue damage and disease. Trends Immunol.23:61-64.

CRIg is a recently discovered complement receptor, which is expressed ona subpopulation of tissue resident macrophages. As a functionalreceptor, the extracellular IgV domain of CRIg is a selective inhibitorof the alternative pathway of complement (Wiesmann et al., Nature,444(7116):217-20, 2006). A soluble form of CRIg has been shown toreverse inflammation and bone loss in experimental models of arthritisby inhibiting the alternative pathway of complement in the joint. It hasalso been shown that the alternative pathway of complement is not onlyrequired for disease induction, but also disease progression. Thus,inhibition of the alternative pathway by CRIg constitutes a promisingtherapeutic avenue for the prevention and treatment of diseases anddisorders the pathogenesis of which involves the alternative pathway ofcomplement. For further details see, e.g. Helmy et al., Cell,125(1):29-32 2006) and Katschke et al., J. Exp Med 204(6):1319-1325(2007).

However, CRIg affinity for the convertase subunit C3b is low (micromolarrange). In order to generate a more potent inhibitor to develop atherapeutic reagent, the crystal structure of CRIg in complex with C3bwas used as a guide and we employed phage display technology to generateCRIg variants with improved binding affinity for C3b.

Thus, the present invention concerns CRIg variants with improvedproperties, such as improved binding affinity for C3b and enhancedinhibitory efficacy.

Identification of Affinity Matured CRIg Variants

As described in greater detail in the Example, phage display of proteinor peptide libraries offers a useful methodology for the selection ofCRIg variants with improved binding affinity for C3b and/or otherimproved properties, such as enhanced biological activity (Smith, G. P.,(1991) Curr. Opin. Biotechnol. 2:668-673). High affinity proteins,displayed in a monovalent fashion as fusions with the M13 gene III coatprotein (Clackson, T., (1994) et al., Trends Biotechnol. 12:173-183),can be identified by cloning and sequencing the corresponding DNApackaged in the phagemid particles after a number of rounds of bindingselection.

Affinity maturation using phage display has been described, for example,in Lowman et al., Biochemistry 30(45): 10832-10838 (1991), see alsoHawkins et al, J. Mol Bio1.254: 889-896 (1992), and in the Examplebelow. While not strictly limited to the following description, thisprocess can be described briefly as: several sites within apredetermined region are mutated to generate all possible amino acidsubstitutions at each site. The antibody mutants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage expressing the various mutants can be cycled through rounds ofbinding selection, followed by isolation and sequencing of those mutantswhich display high affinity. The method is also described in U.S. Pat.No. 5,750,373, issued May 12, 1998.

A modified procedure involving pooled affinity display is described inCunningham, B. C. et al, EMBO J. 13(11), 2508-2515 (1994). The methodprovides a method for selecting novel binding polypeptides comprising:a) constructing a replicable expression vector comprising a first geneencoding a polypeptide, a second gene encoding at least a portion of anatural or wild-type phage coat protein wherein the first and secondgenes are heterologous, and a transcription regulatory element operablylinked to the first and second genes, thereby forming a gene fusionencoding a fusion protein; b) mutating the vector at one or moreselected positions within the first gene thereby forming a family ofrelated plasmids; c) transforming suitable host cells with the plasmids;d) infecting the transformed host cells with a helper phage having agene encoding the phage coat protein; e) culturing the transformedinfected host cells under conditions suitable for forming recombinantphagemid particles containing at least a portion of the plasmid andcapable of transforming the host, the conditions adjusted so that nomore than a minor amount of phagemid particles display more than onecopy of the fusion protein on the surface of the particle; f) contactingthe phagemid particles with a target molecule so that at least a portionof the phagemid particles bind to the target molecule; and g) separatingthe phagemid particles that bind from those that do not. Preferably, themethod further comprises transforming suitable host cells withrecombinant phagemid particles that bind to the target molecule andrepeating steps d) through g) one or more times.

It is noted that, while the CRIg variants of the present invention havebeen identified using phage display, other techniques and other displaytechniques can also be used to identify CRIg variants with improvedproperties, including affinity matured CRIg variants.

The affinity matured CRIg variants of the present invention weredesigned to cover the contact area between CRIg and C3b, which wasidentified using the crystal structure of a CRIg and C3b:CRIg complexdisclosed in U.S. application publication no. 20080045697. I particular,as shown in FIG. 10, libraries 1-5 were designed to cover residuesE8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111, respectively, of thenative sequence full-length CRIg molecule of SEQ ID NO: 2.

In one embodiment, the CRIg variants herein contain an amino acidsubstitution at one or more amino acid positions selected from the groupconsisting of positions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105, and110 in the amino acid sequence of SEQ ID NO: 2.

Representative CRIg variants herein are set forth in FIG. 12.

Preferably, the substitution is at one or more of amino acid positions60, 64, 86, 99, 105 and 110 of the amino acid sequence of full-lengthnative CRIg of SEQ ID NO: 2.

Without limitation, affinity matured CRIg variants specifically includeone or more of the following substitutions within the SEQ ID NO: 2: E8W,W14F, E84Y/W14F; P45F; G42D/D44H/P45F; Q60I; Q64R; Q60I/Q64R; M86Y;M86W, M86F, M86W/Q9R; M86F/Q99R; K110D, K11N; Q105R/K110N; Q105R/K110Q;Q105K/K110D.

Further variants of native sequence CRIg of SEQ ID NO: 2 with two ormore amino acid substitutions are shown in FIG. 12. Specificallyincluded within this group are Q64R/M86Y; Q60I/Q64R/E8Y; Q60I/Q64R/G42D;Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F; Q60I/Q64R/M86Y;Q601/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N; Q60I/Q 105R/K110N;M86Y/E8Y; M86Y/G42D/D44H/P45F; M86Y/P45F; M86Y/G42D/D44H/P45F;M86Y/Q99K/M86Y/Q99R/M86Y/Q105R/M86Y/Q105K/M86Y/Q105R/K110N.

Preferred CRIg variants herein comprise a mutation selected from thegroup consisting of: Q60I; Q64R; Q60I/Q64R; M86Y; Q99L; Q105K/K110D;E8W/Q105R/K110N; Q64R/M86Y; Q60I/Q64R/E8Y; Q60I/Q64R/G42D;Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F; Q60I/Q64R/M86Y;Q60I/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N; M86Y/P45F;M86Y/Q105K.

Particularly preferred variants comprise the mutations Q60I/Q64R/M86Y orQ60I/Q64R/G42D/D44H/P45F.

Variants which contain one or more of the mutations listed above or inFIGS. 12 and 13 but otherwise retain the native CRIg sequence of SEQ IDNO: 2 are specifically included herein. Such variants will be designatedherein by listing the particular mutation followed by “CRIg.” Thus forexample, a variant which differs from native sequence CRIg of SEQ ID NO:2 only by the mutation E8W will be designated as “E8W CRIg,” a variantwhich differs from native sequence CRIg of SEQ ID NO: 2 only by themutations Q60I/Q64R/M86Y will be designated as “Q60I/Q64R/M86Y CRIg,”etc.

Preparation of CRIg Variants

Various techniques are available which may be employed to produce DNA,which can encode proteins for the recombinant synthesis of the CRIgvariants of the invention. For instance, it is possible to derive DNAbased on naturally occurring DNA sequences that encode for changes in anamino acid sequence of the resultant protein. These mutant DNA can beused to obtain the CRIg variants of the present invention. Thesetechniques contemplate, in simplified form, obtaining a gene encoding anative CRIg polypeptide, modifying the genes by recombinant techniquessuch as those discussed below, inserting the genes into an appropriateexpression vector, inserting the vector into an appropriate host cell,culturing the host cell to cause expression of the desired CRIg variant,and purifying the molecule produced thereby.

Somewhat more particularly, a DNA sequence encoding a CRIg variant ofthe present invention is obtained by synthetic construction of the DNAsequence as described in standard textbooks, such as, for example,Sambrook, J. et al., Molecular Cloning (2nd ed.), Cold Spring HarborLaboratory, N.Y., (1989).

a. Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is the preferred method forpreparing substitution, deletion, and insertion variants of a nativeCRIg polypeptide or a fragment thereof. This technique is well known inthe art as described by Zoller et al., Nucleic Acids Res. 10: 6487-6504(1987). Briefly, nucleic acid encoding the starting polypeptide sequenceis altered by hybridizing an oligonucleotide encoding the desiredmutation to a DNA template, where the template is the single-strandedform of the plasmid containing the unaltered or native DNA sequence ofencoding nucleic acid. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template whichwill thus incorporate the oligonucleotide primer, and will code for theselected alteration of starting nucleic acid.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Natl. Acad Sci. USA 75: 5765 (1978).

If phage display is used, the DNA template can only be generated bythose vectors that are either derived from bacteriophage M13 vectors(the commonly available M13 mpl18 and M13 mp19 vectors are suitable), orthose vectors that contain a single-stranded phage origin or replicationas described by Viera et al., Meth. Enzymol. 153:3 (1987). Thus, the DNAthat is to be mutated must be inserted into one of these vectors inorder to generate a single-stranded template. Production of thesingle-stranded template is described in sections 4.21-4.41 of Sambrooket al., supra.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of CRIg, and the other strand (the original template) encodes thenative, unaltered sequence of CRIg. This heteroduplex molecule is thentransformed into a suitable host cell, usually a prokaryote such as E.coli JM-101. After growing the cells, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabelled with³²Phosphate to identify the bacterial colonies that contain the mutatedDNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: Thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (Amersham). This mixture isadded to the template-oligonucleotide complex. Upon addition of DNApolymerase to this mixture, a strand of DNA identical to the templateexcept for the mutated bases is generated. In addition, this new strandof DNA will contain dCTP-(aS) instead of dCTP, which serves to protectit from restriction endonuclease digestion. After the template strand ofthe double-stranded heteroduplex is nicked with an appropriaterestriction enzyme, the template strand can be digested with ExoIIInuclease or another appropriate nuclease past the region that containsthe site(s) to be mutagenized. The reaction is then stopped to leave amolecule that is only partially single-stranded. A completedouble-stranded DNA homoduplex is then formed using DNA polymerase inthe presence of all four deoxyribonucleotide triphosphates, ATP, and DNAligase. This homoduplex molecule can then be transformed into a suitablehost cell such as E. coli JM101, as described above.

Mutants with more than one amino acid to be substituted may be generatedin one of several ways. If the amino acids are located close together inthe polypeptide chain, they may be mutated simultaneously using oneoligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom each other (separated by more than about ten amino acids), it ismore difficult to generate a single oligonucleotide that encodes all ofthe desired changes. Instead, one or two alternative methods may beemployed.

In the first method, a separate oligonucleotide is generated for eachamino acid to be substituted. The oligonucleotides are then annealed tothe single-stranded template DNA simultaneously, and the second strandof DNA that is synthesized from the template will encode all of thedesired amino acid substitutions. The alternative method involves two ormore rounds of mutagenesis to produce the desired mutant. The firstround is as described for the single mutants: wild-type DNA is used forthe template, and oligonucleotide encoding the first desired amino acidsubstitution(s) is annealed to this template, and the heteroduplex DNAmolecule is then generated. The second round of mutagenesis utilizes themutated DNA produced in the first round of mutagenesis as the template.Thus, this template already contains one or more mutations. Theoligonucleotide encoding the additional desired amino acidsubstitution(s) is then annealed to this template, and the resultingstrand of DNA now encodes mutations from both the first and secondrounds of mutagenesis. This resultant DNA can be used as a template in athird round of mutagenesis, and so on.

b. Cassette Mutagenesis

This method is also a preferred method for preparing substitution,deletion, and insertion variants of CRIg. The method is based on thatdescribed by Wells et al. Gene 34:315 (1985). The starting material isthe plasmid (or other vector) comprising gene 1, the gene to be mutated.The codon(s) to be mutated in the nucleic acid encoding the startingCRIg molecule are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in gene 1. After the restriction siteshave been introduced into the plasmid, the plasmid is cut at these sitesto linearize it. A double-stranded oligonucleotide encoding the sequenceof the DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3′ and 5′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence of CRIg.

c. Recombinant Production of CRIg Variants

The DNA encoding variants are then inserted into an appropriate plasmidor vector. The vector is used to transform a host cell. In general,plasmid vectors containing replication and control sequences which arederived from species compatible with the host cell are used inconnection with those hosts. The vector ordinarily carries a replicationsite, as well as sequences which encode proteins that are capable ofproviding phenotypic selection in transformed cells.

For example, E. coli may be transformed using pBR322, a plasmid derivedfrom an E. coli species (Mandel, M. et al., (1970) J. Mol. Biol.53:154). Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3, pDR720, andpPL-X represent expression vectors with the tac, tip, or P_(I) promotersthat are currently available (Pharmacia Biotechnology).

Other preferred vectors can be constructed using standard techniques bycombining the relevant traits of the vectors described herein. Relevanttraits of the vector include the promoter, the ribosome binding site,the variant gene or gene fusion, the signal sequence, the antibioticresistance markers, the copy number, and the appropriate origins ofreplication.

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include cubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serrafia, e.g, Serratiamarcescans, and Shigeila, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X 1776 (ATCC31,537), and E coil W3110 (ATCC 27,325) are suitable. These examples areillustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subloned for growth insuspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for the production of the CRIg variants herein andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

The host cells used to produce the CRIg variants of this invention maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganiccompounds usually present at final concentrations in the micromolarrange), and glucose or an equivalent energy source. Any other necessarysupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture conditions, suchas temperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

When using recombinant techniques, the CRIg variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the CRIg variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed cells, isremoved, for example, by centrifugation or ultrafiltration. Where theCRIg variant is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The CRIg variant prepared from the cells can be purified by knowntechniques, using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and/or affinity chromatography.

Further Modifications of CRIg Variants

The CRIg variants of the present invention may also be modified in a wayto form a chimeric molecule comprising CRIg variant, including fragmentsthereof, fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof CRIg variant, or a fragment thereof, with a tag polypeptide whichprovides an epitope to which an anti-tag antibody can selectively bind.The epitope tag is generally placed at the amino- or carboxyl-terminusof the variant CRIg polypeptide. The presence of such epitope-taggedforms of the CRIg variant can be detected using an antibody against thetag polypeptide. Also, provision of the epitope tag enables the CRIgpolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). Other tag polypeptides include the Flag-peptide (Hopp et al.,BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science, 255:192-194 (1992)); an .quadrature.-tubulin epitopepeptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; andthe T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393-6397 (1990)).

In another embodiment, the chimeric molecule may comprise a fusion ofthe CRIg variant or a fragment thereof with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule. These fusion polypeptides are antibody-like molecules whichcombine the binding specificity of a heterologous protein (an “adhesin”)with the effector functions of immunoglobulin constant domains, and areoften referred to as immunoadhesins. Structurally, the immunoadhesinscomprise a fusion of an amino acid sequence with the desired bindingspecificity which is other than the antigen recognition and binding siteof an antibody (i.e., is “heterologous”), and an immunoglobulin constantdomain sequence. The adhesin part of an immunoadhesin molecule typicallyis a contiguous amino acid sequence comprising at least the binding siteof a receptor or a ligand. The immunoglobulin constant domain sequencein the immunoadhesin may be obtained from any immunoglobulin, such asIgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2),IgE, IgD or IgM.

The simplest and most straightforward immunoadhesin design combines thebinding region(s) of the “adhesin” protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparing theCRIg-immunoglobulin chimeras of the present invention, nucleic acidencoding the extracellular domain of CRIg will be fused C-terminally tonucleic acid encoding the N-terminus of an immunoglobulin constantdomain sequence, however N-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge and CH2 and CH3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of theCRIg-immunoglobulin chimeras.

In some embodiments, the CRIg-immunoglobulin chimeras are assembled asmonomers, or hetero- or homo-multimer, and particularly as dimers ortetramers, essentially as illustrated in WO 91/08298.

In a preferred embodiment, the CRIg extracellular domain sequence isfused to the N-terminus of the C-terminal portion of an antibody (inparticular the Fc domain), containing the effector functions of animmunoglobulin, e.g. immunoglobulin G.sub.1 (IgG 1). It is possible tofuse the entire heavy chain constant region to the CRIg extracellulardomain sequence. However, more preferably, a sequence beginning in thehinge region just upstream of the papain cleavage site (which definesIgG Fc chemically; residue 216, taking the first residue of heavy chainconstant region to be 114, or analogous sites of other immunoglobulins)is used in the fusion. In a particularly preferred embodiment, the CRIgamino acid sequence is fused to the hinge region and CH2 and CH3, or tothe CH1, hinge, CH2 and CH3 domains of an IgG1, gG2, or IgG3 heavychain. The precise site at which the fusion is made is not critical, andthe optimal site can be determined by routine experimentation.

In some embodiments, the CRIg-immunoglobulin chimeras are assembled asmultimer, and particularly as homo-dimers or -tetramers. Generally,these assembled immunoglobulins will have known unit structures. A basicfour chain structural unit is the form in which IgG, IgD, and IgE exist.A four unit is repeated in the higher molecular weight immunoglobulins;IgM generally exists as a pentamer of basic four units held together bydisulfide bonds. IgA globulin, and occasionally IgG globulin, may alsoexist in multimeric form in serum. In the case of multimer, each fourunit may be the same or different.

Alternatively, the CRIg extracellular domain sequence can be insertedbetween immunoglobulin heavy chain and light chain sequences such thatan immunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the CRIg sequence is fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom et al.,Mol. Immunol., 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to aCRIg-immunoglobulin heavy chain fusion polypeptide, or directly fused tothe CRIg extracellular domain. In the former case, DNA encoding animmunoglobulin light chain is typically coexpressed with the DNAencoding the CRIg-immunoglobulin heavy chain fusion protein. Uponsecretion, the hybrid heavy chain and the light chain will be covalentlyassociated to provide an immunoglobulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodssuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

Pharmaceutical Compositions

The CRIg variants of the present invention can be administered for thetreatment of diseases the pathology of which involves the alternativecomplement pathway. Therapeutic formulations are prepared for storage bymixing the active molecule having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine;

preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Lipofections or liposomes can also be used to deliver the polypeptide,antibody, or an antibody fragment, into cells. Where antibody fragmentsare used, the smallest fragment which specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable region sequences of an antibody, peptide molecules can bedesigned which retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology (see, e.g. Marasco et al., Proc. Natl. Acad.Sci. USA 90, 7889-7893 [1993]).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active molecules may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37 C, resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Methods of Treatment

As a result of their ability to inhibit complement activation, inparticular the alternative complement pathway, the CRIg variants of thepresent invention find utility in the prevention and/or treatment ofcomplement-associated diseases and pathological conditions. Suchdiseases and conditions include, without limitation,complement-associated, inflammatory and autoimmune diseases.

Specific examples of complement-associated, inflammatory and immunerelated diseases and disorders that can be targeted by the CRIg variantsherein have been listed earlier.

Further details of the invention are illustrated by the followingnon-limiting Examples.

EXAMPLE 1 Preparation of Affinity Matured CRIg Variants

Materials and Methods

Materials:

Materials—Enzymes and M13-KO7 helper phage (New England Biolabs);Maxisorp immunoplates plates (Nunc. Roskilde, Denmark); 96-well U-bottomPolypropylene plate (COSTAR; Cat. #3365); 96-well flat bottom,non-binding plate (NUNC; Cat. #269620); Horseradish peroxidase/anti-M13antibody conjugate (Pharmacia); 3,3′, 5,5′-Tetramethyl-benzidine, H₂O₂peroxidase substrate (TEB) (Kirkegaard and Perry Laboratories, Inc);Escherichia coli XL1-blue and E. coli. BL21(DE3) (Stratagene); Bovineserum albumin (BSA) and Tween 20 (Sigma); Ni-NTA agarose (Qiagen);Rabbit RBC (Colorado Serum Company; Cat. #CS1081); Gelatin VeronalBuffer (GVB) [100 mL Veronal Buffer (BioWhittaker; Cat. #12-624E);Gelatin (Bovine Skin Type B; SIGMA; Cat. #G9391-100G); C1q-depletedSerum (CompTech; Cat. #A300); fH Protein (Complement Technology; Cat.#A137); Anti-FLAG-HRP, mAb in 50% glycerol, Sigma Cat#A-8592 1.1 mg/mL

Construction of Phage-Display CRIg Libraries

A DNA fragment encoding CRIg was ligated into a XhoI and Spel—digestedphagemid vector (p3DvlzPDZ-gD) (Kunkel et al., Methods Enzymol.154:367-382 (1987)) as wild type control and template for design CRIgvariants. Then, templates with the TAA stop codon at each residuetargeted for randomization were prepared from CJ239 E. coli cells(Kunkel et al., 1987, supra). A soft randomization strategy was used forCRIg variants selection, in which a mutation rate of approximately 50%was introduced at selected position by using a poisoned oligonucletidestrategy with 70-10-10-10 mixtures of bases favoring the wild-typenucleotides. In the libraries design: 5=50% A, 10% G, 10% C and 10% T;6=50% G, 10% A, 10% C and 10% T; 7=50% C, 10% A, 10% G and 10% T; 8=50%T, 10% A, 10% G and 10% C.

Five libraries have been designed.

BCR1, (SEQ ID NO: 7) ATC CTG GAA GTG CAA 656 (SEQ ID NO: 8)AGT GTA ACA GGA CCT 866 (SEQ ID NO: 9) 555 GGG GAT GTG AAT CTTin library 1; BCR2, (SEQ ID NO: 10) AAG TGG CTG GTA CAA 768(SEQ ID NO: 11) 668 TCA 657 775 688 577 ATC TTT (SEQ ID NO: 12)786 CGT 657 TCT TCT GGA GAC CAT in library 2; BCR3, (SEQ ID NO: 13)TTT CTA CGT GAC TCT (SEQ ID NO: 14)877 668 657 757 588 756 756 678 555 TAC 756 GGC CGC CTG CAT GTVGin library 3; BCR4, (SEQ ID NO: 15) CAA TTG AGC ACC CTG (SEQ ID NO: 16)656 586 657 GAC 768 AGC CAC TAC ACG TGT 656 (SEQ ID NO: 17)GTC ACC TGG 756 (SEQ ID NO: 18) ACT CCT GAT GGC AAC in library 4; BCR5,(SEQ ID NO: 19) ACT CCT GAT GGC AAC 756 (SEQ ID NO: 20)GTC 688 768 657 555 ATT ACT GAG CTC CGT in library 5.

Side-directed mutagenesis for the point mutations was carried out asabove by using appropriated codons to produce the respective mutations,and the correct clones were confirmed by sequence.

Library Sorting and Screening to Select CRIg Variants:

Maxisorp immunoplates were coated overnight at 4° C. with C3b (5 μg/ml)and blocked for 1 hr at room temperature with phosphate-buffered saline(PBS) and 0.05% (w/v) bovine serum albumin (BSA). Phage libraries wereadded to the C3b coated plates and incubated at room temperature for 3hr. The plates were washed ten times and bound phage were eluted with 50mM HCl and neutralized with equal volume of 1.0 M Tris base (pH7.5).Recovered phages were amplified by passage through E. coli XL1-blue andwere used for additional rounds of binding selections. After 5 rounds,we select 12 individual clones from each library and grow them in a96-well format in 500 μl of 2YT broth supplemented with carbenicillinand M13-K07 helper phage. Two-fold serial diluted culture supernatantswere added directly in 384 well plates by coated with C3b, anti-gD, BSAand unrelated protein as designed positions. Binding affinity wasmeasured to estimate a phage concentration giving C3b significant higherthan anti-gD but not to BSA and unrelated protein. We fixed the phageconcentration, screening about 200 clones from each library in the sameformat and selected 24-48 clones in which showed significantly to bindto C3b over anti-gD from each library, then sequence them for analysis.

Competitive Phage ELISA

For estimating the binding affinity, a modified phage ELISA was used.The 96 well microtiter plates were coat with 2 ug/m13Cb in 50mMcarbonate buffer (pH9.6) at 4 C over-night. The plates were then blockwith PBS, 0.5% BSA for 1 hour at room temperature. Phage displaying CRIgvariants serially diluted in PBT buffer and binding was measured toestimate a phage concentration giving 50% of the signal at saturation.Subsaturating concentration of phage was fixed and pre-incubated for 2 hwith serial dilutions of C3b, then transferred the mixture to assayplates coated with C3b. After incubating 15 min, the plates were washedwith PBS, 0.05% Tween 20 and incubated 30 min with horseradishperoxidase/anti-M13 antibody conjugate (1:5000 dilution in PBT buffer).The plates were washed, developed with TMB substrate, quenched with 1.0M H₃PO₄, and read spectrophotometrically at 450 nm. The affinity (Ic50)was calculated as the concentration of competing C3b that resulted inhalf-maximal phagemid binding.

Protein Purification

A single colony of E. coli. BL21(DE3) harboring the expression plasmidwas inoculated into 30 mL of LB medium supplemented with 50 μg/mLcarbenicillin (LB/carb medium) and was grown overnight at 37° C. Thebacteria were harvested, washed, resuspended, and inoculated into 500 mLof LB/carb medium. The culture was grown at 37° C. to mid-log phase(A₆₀₀=0.8). Protein expression was induced with 0.4 mM isopropyl1-thio-D-galactopyranoside, and the culture was grown for 24 h at 30° C.The bacteria were pelleted by centrifugation at 4000 g for 15 min,washed twice with phosphate-buffered saline (PBS), and frozen for 8 h at−80° C. The pellet was resuspended in 50 mL of PBS, and the bacteriawere lysed bypassing through the Microfluidizer Processing or sonicateequipments. The CRIg variant proteins were purified with 2 ml NI-NTAagarose and gel filtration.

mutCRlg-huFc Fluid Phase Competitive Binding ELISA:

huCRIg(L)-LFH was diluted to 2μg/mL in PBS, pH 7.4, and coated ontoMaxisorp 384-well flat bottom plates (Nunc, Neptune, NJ) by incubatingovernight (16-18 hr) at 4° C. (25 ul/well). The plates were washed 3times in Wash Buffer (PBS, pH7.4, 0.05% Tween 20), and 50 ul/well ofBlock Buffer (PBS, pH 7.4, 0.5% BSA) was added to each well. The plateswere allowed to block for 1-3hr; this and all subsequent incubationswere performed on an orbital shaker at room temperature. During theblocking step, C3b (purified at Genentech) was diluted to 20 nM in AssayBuffer (PBS pH7.4, 0.5% BSA, 0.05% Tween-20), and the mutCRlg-huFcmolecules were serially diluted in Assay Buffer, over a concentrationrange of 20,000—0.34 nM. The C3b and mutCRlg-huFc molecules were thenmixed 1:1 and allowed to pre-incubate for 0.5-1 hr. The blocked plateswere washed three times (as described above), and the C3b:mutCRlg-huFccomplexes were added to the reaction plates (25 ul/well). After a 1-2 hrincubation, The ELISA plates were washed three times, (as describedabove) and plate-bound C3b was detected by the addition of an anti-humanC3b antibody (clone 5F202, US Biological, Swampscott, Mass.; 600 ng/mL,25 ul/well). The plates were incubated for 1-2 hr and washed asdescribed above. HRP-conjugated anti-murine Fc IgG (JacksonImmunoResearch, West Grove, Pa.) diluted 1:2,000 was then added (25ul/well), and the plates were incubated for 1-2 hr. After a final wash,25 ul/well of TMB substrate (Kirkegaard & Perry Laboratories,Gaithersburg, Md.) was added to the ELISA plates. Color development wasstopped after approximately 8min by adding 25 ul/well 1.0M phosphoricacid. Absorbance at 450 nm and 650 nm was determined using a SpectraMax250 microtiter plate reader (Molecular Devices, Sunnyvale, Calif.).

Complement Activation Assay:

The ability of mutCRlg-Fc to inhibit complement activation was evaluatedusing the Wieslab™ Complement System Alternative Pathway Kit (AlpcoDiagnostics, Salem, N.H.). Serially diluted mutCRlg-Fc (400 to 0.2 nM)and C1q deficient human serum (5%) (Complement Technology, Tyler, Tex.)were prepared at twice the final desired concentration, mixed 1:1, andpre-incubated for 5 min on an orbital shaker at 300RPM prior to addingto the LPS-coated ELISA plates (100 ul/well). The remainder of the assaywas following manufacturer's instructions. Briefly, the samples in theELISA plates were incubated for 60-70 min at 37° C. and then washedthree times in Wash Buffer (PBS, pH7.4, 0.05% Tween 20). 100 ul/well ofthe anti-C5b-9 conjugate was added to the ELISA plate. After a 30 minincubation at room temperature, the ELISA plate was washed as describedabove, and 100 ul of substrate was added per well, and the plates wereincubated at room temperature for an additional 30 min. The colordevelopment was stopped by adding 50 ul/well of 5 mM EDTA. Absorbance at405 nm was determined using a MultiSkan Ascent microtiter plate reader(Thermo Fisher Scientific, Milford, Mass.).

Hemolysis Inhibition Assay:

Rabbit red blood cells (Colorado Serum Company, Denver, Colo.) werewashed three times with Veronal Buffer (Sigma, St. Louis, Mo.)containing 0.1% bovine skin gelatin (Sigma) (GVB), centrifuging at 1500rpm, 4° C. for 10 minutes for each wash. After the final centrifugationstep, the cells were resuspended in GVB at a final concentration of2×10⁹ cells/mL. Complement inhibitors serially diluted in GVB were addedto 96-well U-bottom polypropylene plate(s) (Costar, Cambridge, Mass.) at50 μL/well followed by 20μL/well of rabbit red blood cells diluted 1:2in 0.1M MgCl₂/0.1M EGTA/GVB. The in-plate complement cascade wastriggered by the addition of 30 μL/well C1q-depleted serum (ComplementTechnology, Tyler, Tex.), pre-diluted 1:3 with GVB. The plate(s) wereincubated with gentle agitation for 30 minutes at room temperaturebefore stopping the reaction with 100 μL/well 10 mM EDTA/GVB. Aftercentrifuging the plate(s) at 1500 rpm for 5 minutes, the supernatantswere transferred to clear flat bottom, non-binding, 96-well plate(s)(Nunc, Neptune, N.J.) and the optical densities were read at 412 nmusing a microplate reader (Molecular Devices, Sunnyvale, Calif.).

Alpha Screen Competitive Assay:

The potential cross-reacivity of the mutant CRIg molecules to C3 wasevaluated using the AlphaScreen® Histidine (Nickel Chelate) DetectionKit (PerkinElmer, Waltham, Mass.). Serially diluted human C3 and C3b(3,000 to 0.7 nM), as well as fixed concentrations of biotinylated iC3b(30 nM), and both mutant CRIg (mutCRlg) and wild-type CRIg molecules(15-60 nM) were prepared at three times the final desired concentration,mixed 1:1:1, and pre-incubated at ambient temperature for 30 minutes onan orbital shaker at 3000 TPM. A 1:1 mixture of streptavidin donor beadsand nickel chelate acceptor beads (0.1 mg/mL each) was prepared at fourtimes the final desired concentration and added to the reaction. Thereaction plate was incubated at ambient temperature for 60 minutes on anorbital shaker at 3000 rpm protected from light. The plate was analyzedon an AlphaQuest®-HTS microplate analyzer (PerkinElmer, Waltham, Mass.).

Surface Plasmon Resonance

Affinities of C3b for mutant and wild-type CRIg were determined by usingsurface plasmon resonance measurements on a Biacore® A100 instrument (GEhealthcare). An anti-Fc capture format was employed and the K_(D) wascalculated from equilibrium binding measurements. The sensor chip wasprepared using the anti-muFc capture kit (BR-1008-38) followinginstructions supplied by the manufacturer. Mutant or wild-type CRIg wasdiluted in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.01%Tween-20) to 1 μg/mL and injections of 60 μL were made such that ˜100 RUof fusion protein were captured on one spot of the chip surface.Sensorgrams were recorded for 10 min injections of solutions of variedC3b concentration over the CRIg spot with subtraction of signal for areference spot containing the capture antibody but no CRIg. Data wereobtained for a 2-fold dilution series of C3b ranging in concentrationfrom 4 μM to 15.6 nM with the flow rate at 10 μL/min and at atemperature of 25° C. The surface was regenerated between binding cyclesby a 30 second injection of 10 mM Gly-HCl pH 1.7. Plateau valuesobtained at the end of each C3b injection were used to calculate K_(D)using the Affinity algorithm of the Biacore A100 Evaluation Softwarev1.1 (Safsten et al. (2006) Anal. Biochem. 353:181).

Results

Phage Library Design

We used the crystal structure of CRIg in complex with C3b to designtarget libraries. Five libraries were designed to cover the contact areabetween CRIg and C3b (FIG. 4). CRIg libraries were constructed as afusion to the g3p minor coat protein in a monovalent phage displayvector (Zhang et al., J Biol Chem 281(31): 22299-311 (2006)). Weintroduced stop codons by mutagenesis into the CRIg-coding portion ofthe phage plasmid at each residue to be randomized. Each constructcontaining a stop-codon was then used to generate the phage-displaylibrary (see material and method). A “soft randomization” strategy wasused to select binders to maintain a wild-type sequence bias such thatthe selected positions were mutated only 50% of the time. All fivelibraries were obtained with an average diversity of >10¹⁰ independentsequences per library. (FIG. 10).

Selections with CRIg Phage Library

Following four rounds of binding selection, we obtained 38 unique clonesfrom thes five libraries. (FIG. 11). In library 1, lysine at position 15was conserved. Aromatic residues, tyrosine and tryptophan, replacedglutamic acid at position 8. Position 14 was occupied by either theparental tryptophan or a homologous phenylalanine In library 2, wesequenced 24 clones and all of them revealed consensus. Position 42, 46and 47 were conserved as wild type. Asparagin, histidine andphenylalanine replaced the wild type sequence at position 43, 44 and 45.In library 3, we randomized 10 positions and the sequences exhibitedcomplete conservation at position 54, 55, 56, 57, 58, 61, 62 and 63.Isoleucine or lysine was occupied at position 60. At position 64,glutamine was replaced by arginine or conserved. In library 4, aromaticresidues dominated at position 86 and homologous basic residues,arginine and lysine, dominated at position 99. Position 85, 87 and 95were also soft randomized, but appeared highly conserved. In library 5,two significant homologous basic residues, lysine and arginine werepreferred over glutamine at position 105. Negatively charged residues,aspartic acid or acidic residues, asparagine was dominated at position110.

We estimated the affinities of some of the mutants by competitive phageELISA (data not shown), and we found that there were clones in library 3which were approximately eightfold times better C3b binders than wildtype CRIg.

Determination of in vitro Binding Affinity and in vivo BiologicalPotency

In order to identify critical residues for increasing the bindingaffinity to C3b and potency in hemolytic inhibition assay, the nextapproach was to design second generation of CRIg variants byincorporating dominant single mutation and keep other positions as wildtype, or choosing 2-3 high-affinity clones from first generationphage-libraries which were determined by phage ELISA. In order toaccurately measure the affinity and potency of our mutants, we expressedall the variants as isolated proteins. The results (FIG. 12) fromhemolytic inhibition assay showed that L12 from library 1, L33 fromlibrary 3 and L41 from library 4 significantly increased the potency by4 to 10 fold compare to wild type in a hemolytic assay. L32 from library3 showed a 10 fold improved IC50 compared to wild type CRIg. The dataalso demonstrated that the binding affinity and the potency from thecell-based assays were not correlated.

Combination of Mutants

Based on the results from the second generation labraries, we designedthe third generation of mutants in order to further improve the potencyin the hemolytic assay and binding affinity. We chose three of thebiologically most potent mutants (L12-8W, L33-Q60I/L32-Q64R andL41-M86Y) and one of the highest binding affinity mutant (L32-Q64R) as atemplate. Then we combined these mutants with other biological potentclones obtained in the second generation of libraries to determine anoptimal set of mutations that increase potency in the hemolytic assayand binding affinity. We expressed and purified the CRIg variants fordetailed analysis. The data showed (FIG. 13) that the combo mutants fromL12 didn't improve inhibition potency and even displayed a loweractivity compare with the parent mutant despite a 3-6 fold higherbinding affinity of the WL41 and WL59 mutants. Within the mutants fromL32, RL41 demonstrated a 1.8 fold better binding affinity than wild typeand a 6 fold better potency in the hemolytic assay. All the mutants fromL33 group showed the significant increased binding affinity; about a27-226 fold increase compared with wild type although the potency in thehemolysis assay did not increase significantly. We also noticed that60I-64R and 86Y was involved in most the affinity improved combo clones.

Improved Binding Affinity and Complement Inhibitory Activity of CRIgQ64R M86Y mutant

We selected mutant Q64R M86Y, which had the highest affinity in thecompetitive ELISA (FIG. 4A and FIG. 12) for further analysis. In orderto determine the binding affinity of CRIg wt and CRIg Q64R M86Y for C3b,Biacore analysis of CRIg wt and CRIg Q64R M86Y was performed. Theaffinity of CRIg Q64R M86Y was improved 5 fold over wildtype CRIg (FIGS.6A and 6B). Previous studies have shown that CRIg wt selectively bindsto C3b but not to native C3 (Wiesman et al., Nature, 444(7116):217-20,2006). Since mutagenesis may change this selectivity we compared theaffinity of CRIg Q64R M86Y for C3b versus C3 in an alpha-screenfluid-phase competitive assay. CRIg Q64R M86Y competed with soluble C3b,but not with soluble C3, indicating that mutagenesis did not affect theselectivity of CRIg for the active component C3b (FIG. 7). Thisselectivity was further confirmed by analysis of these residues in thestructure of CRIg Q64R M86Y in complex with C3b (data not shown).

To test whether the improved affinity and conserved selectivity for C3btranslates into improved efficacy, we tested CRIg Q64R M86Y versus CRIgwt in an erythrocyte-based hemolytic assay selective for the alternativepathway of complement. CRIg Q64R M86Y showed a 4-fold improved IC50 ascompared to CRIg wt (FIG. 8A). To further substantiate improved potencytoward alternative pathway complement inhibiton, we compared inhibitoryactivity of CRIg Q64R M86Y with CRIg wt in a LPS-based assay selectivefor the alternative pathway of complement. Here, CRIg showed a 180-foldimprovement in IC50 as compared to the wildtype recombinant protein.CRIg wt and CRIg Q64R M86Y did not affect complement activation throughthe classical pathway. Thus, a two amino acid substitution in theCRIg-C3b binding interface results in a molecule with improved bindingaffinity and superior complement inhibitory activity in two differentassays with selectivity for the alternative pathway of complement.

To further determine whether increased binding affinity and potencytranslate into improved therapeutic efficacy, we compared the protectiveeffect of wt and Q64R M86Y version of CRIg in a serum-transfer model ofarthritis. Previous studies have shown that CRIg potently inhibitsinflammation and bone destruction in collagen- and antibody-inducedarthritis (Katschke et al., J. Exp Med 204(6):1319-1325 (2007)).

Here, CRIg efficacy was tested in a third preclinical model of immunecomplex-mediated arthritis. A spontaneous murine model of rheumatoidarthritis, K/BxN, mimics many of the clinical and histologic features ofhuman disease with arthritis. Mice were injected with 50 microliterserum obtained from K/BxN mice on day 0. Animals were checked daily andthe extent of disease was scored by visual observation. All mice weresacrificed on day 6.

Mice were injected subcutaneously with indicated amount of eitherisotype control or hCRIg-mIgG1 or hCRIg-RL41-mIgG1 recombinant proteinsdaily in 100 ul sterile saline starting on day −1.

Monitoring and Scoring:

Score for each paw.

-   0=No evidence of erythema and swelling-   1=Erythema and mild swelling confined to the mid-foot (tarsal) or    ankle-   2=Erythema and mild swelling extending from the ankle to the    mid-foot-   3=Erythema and moderate swelling extending from the ankle to the    metatarsal joints-   4=Erythema and severe swelling encompass the ankle, foot and digits

Mean score=sum of the 4 paw scores.

Disease stages, mild (mean score 1-3), moderate (mean score 4-8) andsevere disease (mean score 9-above). The mean score reflects the numberof joints involved.

On day 6, blood sample were collected by intracardiac puncture underanesthesia before sacrifice. The amount of hCRIg-Fc fusion proteins willbe measured using the serum. Joints were collected for histologyevaluation.

Transfer of serum from KRN mice into Balb/c recipients results in arapid and robust immune response characterized by symmetric inflammationof the joints. Arthritis induction is mediated by antiGlucose-6-phosphate isomerase autoantibodies that form pro-inflammatoryimmune complexes in the joints (Kouskoff, V., Korganow, A. S.,Duchatelle, V., Degott, C., Benoist, C., and Mathis, D. (1996).Organ-specific disease provoked by systemic autoimmunity. Cell 87,811-822.) Development of arthritis is fully dependent on an intactalternative complement pathway and on Fc receptor function as shown bythe lack of disease in mice deficient in alternative pathway complementcomponents and in mice deficient in the common fc-receptor gamma chain(Ji, H., Ohmura, K., Mahmood, U., Lee, D. M., Hofhuis, F. M., Boackle,S. A., Takahashi, K., Holers, V. M., Walport, M., Gerard, C., et al.(2002). Arthritis critically dependent on innate immune system players.Immunity 16, 157-168.) Due to the rapid onset and severity of disease,treatment with CRIg wt-Fc fusion protein reduced arthritis scores byonly 22% (FIGS. 9A-B). Treatment with CRIg Q64R M86Y showed a reductionin arthritis scores by 66%. Histological examination showed asignificant reduction in infiltration of immune cells consistingprimarily of neutrophils and macrophages in CRIg Q64R M86Y treated miceversus CRIg wt or control fusion protein-treated mice (FIGS. 9C-D).Serum concentrations of CRIg wt and CRIg Q64R M86Y were similarindicating that the difference in arthritis scores was not due to adifference of halflife of the CRIg wt versus CRIg Q64R M86Y protein.Thus, we show that increased binding affinity of CRIg to its target C3btranslates into a significantly improved therapeutic efficacy.

All patent and literature references cited in the present specificationare hereby expressly incorporated by reference in their entirety.

While the present invention has been described with reference to whatare considered to be the specific embodiments, it is to be understoodthat the invention is not limited to such embodiments. To the contrary,the invention is intended to cover various modifications and equivalentsincluded within the spirit and scope of the appended claims.

What is claimed is:
 1. A method for the prevention or treatment of acomplement-associated disease or condition, comprising administering toa subject in need of such treatment a prophylactically ortherapeutically effective amount of a CRIg variant comprising an aminoacid substitution in a region selected from the group consisting ofE8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111 of the amino acidsequence of SEQ ID NO: 68, or an immunoadhesin comprising such variant.2. The method of claim 1, wherein said variant selectively binds to C3bover C3, or a fragment thereof
 3. The method of claim 1, wherein saidvariant has increased binding affinity to C3b over native sequence humanCRIg of SEQ ID NO:
 2. 4. The method of claim 3, wherein the bindingaffinity is increased by at least 2 fold, or by at least 5 fold, or byat least 10 fold, or by at least 90 fold.
 5. The method of claim 1,wherein said variant is more potent inhibitor of the alternativecomplement pathway than native sequence human CRIg of SEQ ID NO:
 2. 6.The method of claim 5, wherein said variant is at least 2-time morepotent, or at least 5-time more potent, or at least 10-times more potentthan native sequence human CRIg of SEQ ID NO:
 2. 7. The method of claim1, wherein said variant comprises an amino acid substitution at one ormore amino acid positions selected from the group consisting ofpositions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105,and 110 n the aminoacid sequence of SEQ ID NO:
 2. 8. The method of claim 1, wherein sa idvariant comprises an amino acid substitution at one or more of aminoacid positions 64, 86, 99, 105 and 110 in the amino acid sequence of SEQID NO:
 2. 9. The method of claim 1, wherein said variant comprises oneor more substitutions selected from the group consisting of E8W, W14F,E84Y/W14F; P45F; G42D/D44H/P45F; Q60I; Q64R; Q60I/Q64R; M86Y; M86W,M86F, M86W/Q9R; M86F/Q99R; K110D, K11N; Q105R/K110N; Q105R/K110Q; andQ105K/K110D.
 10. The method of claim 1, wherein said variant comprisesone or more substitutions selected from the group consisting ofQ64R/M86Y; Q601/Q64R/E8Y; Q60I/Q64R/G42D; Q60I/Q64R/P45F;Q60I/Q64R/G42D/D44H/P45F; Q60I/Q64R/M86Y; Q60I/Q64R/Q105R;Q60I/Q64R/Q105K; Q60I/Q64R/K110N; Q60I/Q105R/K110N; M86Y/E8Y;M86Y/G42D/D44H/P45F; M86Y/P45F; M86Y/G42D/D44H/P45F; andM86Y/Q99K/M86Y/Q99R/M86Y/Q105R/M86Y/Q105K/M86Y/Q105R/K110N.
 11. Themethod of claim 1, wherein said variant comprises one or moresubstitutions selected from the group consisting of Q60I; Q64R;Q60I/Q64R; M86Y; Q99L; Q105K/K110D; E8W/Q105R/K110N; Q64R/M86Y;Q60I/Q64R/E8Y; Q60I/Q64R/G42D; Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F;Q60I/Q64R/M86Y; Q60I/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N;M86Y/P45F; and M86Y/Q105K.
 12. The method of claim 1, wherein saidvariant comprises a Q60I/Q64R/M86Y or Q60I/Q64R/G42D/D44H/P45Fsubstitution.
 13. The method of claim 1, wherein said variant is shorterthan the mature full-length CRIg of SEQ ID NO:
 68. 14. The method ofclaim 1, wherein said immunoadhesin comprises the extracellular domainof said CRIg variant.
 15. The method of claim 1, wherein saidcomplement-associated disease is an inflammatory disease or anautoimmune disease.
 16. The method of claim 15, wherein saidcomplement-associated disease is selected from the group consisting ofrheumatoid arthritis (RA), adult respiratory distress syndrome (ARDS),remote tissue injury after ischemia and reperfusion, complementactivation during cardiopulmonary bypass surgery, dematomyositis,pemphigus, lupus nephritis and sesultant glomerulonephritis andvasculitis, cardiopulmonary bypass, cardioplegia-induced coronaryendothelial dysfunction, type II membranoproliferativeglomerulonephritis, IgA nephropathy, acute renal failure,cryoglobulemia, antiphospholipid syndrom, age-related maculardegeneration, uveitis, diabetic retinopathy, allo-transplantation,hyperacute rejection, hemodialysis, chronic occlusive pulmonary distresssyndrome (COPD), asthma, aspiration pneumonia, utricaria, chronicidiopathic utricaria, hemolytic uremic syndrome, endometriosis,cardiogenic shock, ischemia reperfusion injury, and multiple schlerosis(MS).
 17. The method of claim 15, wherein said complement-associateddisease is selected from the group consisting of inflammatory boweldisease (IBD), systemic lupus erythematosus, rheumatoid arthritis,juvenile chronic arthritis, spondyloarthropaties, systemic sclerosis(scleoderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic polyneuropathy, hepatobiliarydiseases such as infectious hepatitis (hepatitis A, B, C, D, E and othernonhepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases (e.g., cystic fibrosis),gluten-sensitive enteropathy, Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases of thelung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection, graft-versus host disease, Alzheimer'sdisease, paroxysmal nocturnal hemoglobinurea, hereditary angioedema,atherosclerosis and type II membranoproliferative glomerulonephritis.18. The method of claim 15, wherein said complement-associated diseaseis rheumatoid arthritis (RA).
 19. The method of claim 15, wherein saidcomplement-associated disease is a complement-associated eye condition.20. The method of claim 19, wherein said complement-associated eyecondition is selected from the group consisting of all stages ofage-related macular degeneration (AMD), uveitis, diabitic and otherischemia-related retinopathies, endophthalmitis, and other intraocularneovascular diseases.
 21. The method of claim 20 wherein the intraocularneovascular disease is selected from the group consisting of diabeticmacular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO),corneal neovascularization, and retinal neovascularization.
 22. Themethod of claim 19 wherein said complement-associated eye condition isselected from the group consisting of age-related macular degeneration(AMD), choroidal neovascularization (CNV), diabetic retinopathy (DR),and endophthalmitis.
 23. The method of claim 22 wherein said AMD is wetAMD.
 24. The method of claim 23 wherein said AMD dry or atrophic AMD.25. The method of claim 1 wherein said subject is a mammal.
 26. Themethod of claim 25 wherein said mammal is a human.
 27. A method forinhibition of the production of C3b complement fragment in a mammalcomprising administering to said mammal an effective amount of a CRIgvariant comprising an amino acid substitution in a region selected fromthe group consisting of E8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111of the amino acid sequence of SEQ ID NO: 68, or an immunoadhesincomprising said variant.